Interrogation of novel pathways regulating VLDL production and plasma lipids

描述(申请人提供)：含载脂蛋白B的脂蛋白(apoB-LP)是冠状动脉疾病(CAD)的重要致病危险因素。载脂蛋白B-Lp升高的最常见原因是肝脏过度产生极低密度脂蛋白，但调节肝脏极低密度脂蛋白组装和分泌的分子过程仍不完全清楚。人类遗传学已经确定了与血浆apoB-LP水平以及与冠心病密切相关的以前未被怀疑的基因。与apoB-LP和CAD相关的两个最引人注目的新基因是SORT1和TRIB1，前者编码一种名为sortilin的蛋白质，另一种编码一种名为tribble-1的蛋白质。我们最近发表了对SORT1基因座的研究，在小鼠身上证明了山梨素在肝脏中的过度表达降低了VLDL分泌和低密度脂蛋白水平，而敲除山梨素则增加了VLDL分泌和低密度脂蛋白水平。同时，我们对TRIB1基因座进行了研究，发现TRIBBLES-1过表达降低了VLDL的分泌，而TRIB1基因敲除的小鼠增加了VLDL的分泌。因此，这两个基因座都编码调节肝脏产生极低密度脂蛋白的蛋白质。然而，它们影响这一复杂过程的分子机制仍不清楚。这项建议的目标是进行一系列详细的实验，旨在阐明这些蛋白质调节肝脏VLDL组装和分泌的分子和细胞机制。关于山梨素，我们将验证这样的假设，即山梨素作为一种分类蛋白，将高尔基体中的新生极低密度脂蛋白结合和分类到溶酶体上，使其从分泌途径分流。关于TRIBBLES-1，我们将检验TRIBBLES-1调节肝细胞中MAP激酶活性的假设，以及TRIBBLES-1靶向C/EBP1和/或2在肝细胞中翻译后降解的假设。我们将在肝细胞培养系统以及山梨素和TRIBBLES-1功能获得和丧失的小鼠身上进行实验。此外，我们将测试SORT1和TRIB1中自然发生的突变对功能的影响。拟议目标的成功完成有望为调节人类极低密度脂蛋白的产生从而调节血浆脂质的新生物途径提供新的见解，并可能为减少极低密度脂蛋白的产生和心血管风险提供新的治疗目标。

DESCRIPTION (provided by applicant): Lipoproteins containing apolipoprotein B (apoB-LP) are important causal risk factors for coronary artery disease (CAD). The most common cause of elevated apoB-LP is overproduction by the liver of VLDL, but the molecular processes regulating hepatic VLDL assembly and secretion remain incompletely understood. Human genetics has identified previously unsuspected genes that are strongly associated with plasma levels of apoB-LP as well as with CAD. Two of the most compelling new genes associated with apoB-LP and CAD are SORT1, encoding a protein called sortilin, and TRIB1, encoding a protein known as tribbles-1. We recently published studies of the SORT1 locus, demonstrating in mice that overexpression of sortilin in the liver reduces VLDL secretion and LDL-C levels whereas knockdown of sortilin increases VLDL secretion and LDL-C levels. In parallel, we investigated the TRIB1 locus, and found that overexpression of tribbles-1 reduced VLDL secretion and Trib1 knockout mice had increased VLDL secretion. Thus, both of these loci encode proteins that regulate the hepatic production of VLDL. However, the molecular mechanisms by which they influence this complex process remain unknown. The goal of this proposal is to perform a series of detailed experiments intended to elucidate the molecular and cellular mechanisms by which these proteins modulate hepatic VLDL assembly and secretion. With regard to sortilin, we will test the hypothesis that sortilin acts as a sorting protein to bind and sort nascent VLDL from Golgi to lysosome, diverting it from the secretory pathway. With regard to tribbles-1, we will test the hypotheses that tribbles-1 regulates MAP kinase activity in hepatocytes, and that tribbles-1 targets C/EBP1 and/or 2 for post-translational degradation in hepatocytes. We will perform experiments in hepatocyte cell culture systems, as well as in mice with gain and loss of function of sortilin and tribbles-1. In addition, we will test naturally-occurring mutations in SORT1 and TRIB1 for functional effects. Successful completion of the proposed aims promises to yield novel insights regarding new biological pathways regulating VLDL production and thus plasma lipids in humans, and could provide novel therapeutic targets for reducing VLDL production and cardiovascular risk.